The long-term objective of this research proposal is to determine the biological function of gamma-delta T cells in man. The gamma-delta repertoire in humans is biased towards cells bearing a T cell receptor (TCR) encoded by a single set of variable (V) genes (V-gamma9 and V- delta2). Studies of gamma-delta T cells in mice have suggested that targeted V-gene rearrangement, stochastic events and thymic selection may account for the highly ordered production of subtypes of gamma-delta T cells. However, several striking differences in gamma-delta cell biology in rodents and man suggest that the results from studies of gamma-delta T cell ontogeny carried out in mice cannot be extrapolated to gamma-delta T cell development and function in man. Based upon our own preliminary studies we are proposing that the gamma-delta T cell repertoire of adults is generated as a result of selection events that occur, at least in part, in tissues outside the thymus during fetal development. Once formed, these cells function primarily as part of the fetal immune system. This hypotheses implies, therefore, that the ontogeny of the V-gamma9/V- delta2+gamma-delta repertoire is intimately related to their specialized function. To test this hypothesis, we propose to perform studies examining the development and specificity of human V-gamma9/V-delta2+ gamma-delta T cells which have the following specific aims: 1. To identify at what stage in gestation and in which fetal tissues V-gamma9/V-delta2+ gamma-delta T cells are first generated. In particular, we will determine whether this population of cells is thymically- or extrathymically-derived and, if fetal-derived gamma-delta T cell populations are part of the adult repertoire. 2. To identify the ligand(s) recognized by a V-gamma9/V- delta2-TCR expressed by a population of gamma-delta T cells that arise, and predominate, during normal fetal development. To detect and visualize cells in situ, in fetal tissues, that express the genes encoding the V-gamma9/V-delta2-TCR, sensitive polymerase chain reaction (PCR)/Southern blotting and in situ hybridization assays will be used, respectively. DNA-sequencing will be used to authenticate PCR- amplified TCRs, to determine the structural diversity and, identify unique sequences that distinguish the repertoire of cells that express this receptor in different tissues throughout fetal development. An immunomagnetic separation procedure will facilitate the isolation of this population of cells, allowing TCRgamma-delta gene expression to be correlated with receptor protein expression and for their phenotypic characterization. The antigen(s) reactive with a V-gamma9/V-delta2 TCR of fetal T cells will be identified and subsequently characterized on the basis of their reactivity with either an intact, assembled gamma-deltaTCR in soluble form, or the cell-associated form expressed by fetal gamma- delta T cell clones. Defining the origin of human gamma-delta T cells (specific aim 1) and the molecular interactions involved in their generation (specific aim 2) are not only essential for determining the nature of antigens recognized by gamma-deltaTCR and the function of human gamma-delta T cells but also for understanding the mechanisms that operate to maintain self-tolerance within the gamma-delta T cell subset. This is of particular importance in light of recent clinical studies demonstrating the involvement of gamma-delta T cells in the pathogenesis of certain autoimmune disorders.